Compositions and methods for screening for Lyme disease

ABSTRACT

The invention provides compositions, methods, and kits for the diagnosis or detection of infection by a pathogen that causes Lyme disease in a subject.

INCORPORATION BY REFERENCE

This application is a 35 U.S.C §371 U.S. national entry of IntenationalApplication PCT/US2011/027888(WO 2011/112805) having an Internationalfiling date of Mar. 10, 2011, which claims the benefit of U.S.Provisional Application Ser.No. 61/312,520, filed Mar.10, 2010, thecontents of which are incorporated herein by reference in theirentirety.

This application claims the benefit of U.S. Provisional Application Ser.No. 61/312,520 filed Mar. 10, 2010, the contents of which isincorporated herein by reference in its entirety.

SEQUENCE LITSTIONG

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 28, 2014, isnamed 85313US(47992)_SL.txt and is 33,555 bytes in size.

GOVERNMENT SUPPORT

This work was supported by the Intramural Research Program of theNational Institutes of Health, the National Institute of Dental andCraniofacial Research, the NIH Clinical Center, and the NationalInstitute for Allergy and Infectious Disease. The Government has certainrights to this invention.

BACKGROUND

Lyme disease is caused by the spirochete Borrelia burgdorferi (Bb) inNorth America and predominantly by Borrelia afzelii and Borrelia gariniiin Eurasia. The pathogen is transmitted by the bite of a tick (Ixodessp.), deer tick or western black-legged tick in North America. One ofthe typical first signs of Borrelia sp. infection is erythema migrans(EM), a bull's eye-like skin lesion, which arises within a few days ofthe bite. However, the bite may not be painful, and a rash does notdevelop in all patients. Patients can also experience flu-like symptoms,however, such symptoms may be attributed by the patient to any of anumber of other causes.

After infection, the spirochetes can disseminate in the bloodstream tovarious target tissues. Prompt treatment with antibiotics can typicallykill the pathogen and prevent long term ill effects of infection. If theinfection is not treated, or not successfully treated, it can result inneurological, rheumatological, and cardiac damage over time. Some commonsymptoms from long-term Lyme infection include arthritis, facial palsy,and neuroboreliosis. Even after antibiotic treatment of Lyme disease,some individuals show post-Lyme disease syndrome (PLDS) and havelingering symptoms such as fatigue, musculoskeletal pain, and cognitivecomplaints.

Because of the difficulty in culturing Borrelia bacteria in thelaboratory, diagnosis of Lyme disease is typically based on clinicalexam findings and a history of exposure to endemic Lyme areas (Ryan K J,Ray C G (editors) 2004. Sherris Medical Microbiology (4th ed.). McGrawHill. pp. 434-437). The EM rash, which does not occur in all cases, isconsidered sufficient to establish a diagnosis of Lyme disease even whenserologic blood tests are negative (Hofmann et al., 1996, Infection 24:470-472.; Pachner et al., 1989. Rev. Infect. Dis. 11 Suppl 6:S1482-1486). Serological testing can be used to support a clinicallysuspected case but is not diagnostic by itself (Ryan K J, Ray C G(editors) 2004. Sherris Medical Microbiology (4th ed.). McGraw Hill. pp.434-437).

SUMMARY OF THE INVENTION

The invention provides compositions, methods, and kits for the detectionof infection by a Borrelia sp. pathogen.

The invention provides compositions containing an isolated peptidehaving an amino acid sequence at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to SEQ IDNO: 1, wherein a 10-fold molar excess of the peptide inhibits binding ofat least 50% of a peptide comprising the amino acid sequence of SEQ IDNO: 1 to a sample from a subject suffering from an infection by Borreliaburgdorferi. In certain embodiments, the isolated peptide has the aminoacid sequence of SEQ ID NO: 1. A sample from a subject suffering frominfection by Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii,and/or Borrelia valaisiana can be evaluated using these compositions inroutine diagnostic methods such as those provided herein. Inembodiments, the subject is suffering from Borrelia burgdorferi.

The invention provides compositions that include the mixtures of theantigenic components of SEQ ID NO: 1. Such compositions according to theinvention contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc)isolated peptides, wherein the peptides include amino acid sequences ofSEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 9. The mixtures can includeseparate peptides having the sequences of SEQ ID NO: 4, SEQ ID NO: 5,and SEQ ID NO: 9. The peptides can have sequences of SEQ ID NO: 4, SEQID NO: 5, and SEQ ID NO: 9 joined, for example, by covalent linkage,such as a peptide bond or a peptide linker. A sample from a subjectsuffering from infection by Borrelia burgdorferi, Borrelia afzelii,and/or Borrelia garinii can be evaluated using these compositions inroutine diagnostic methods such as those provided herein. Inembodiments, the subject is suffering from Borrelia burgdorferi.

The invention provides compositions containing an isolated peptidehaving an amino acid sequence at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to SEQ IDNO: 3, wherein a 10-fold molar excess of the peptide inhibits binding ofat least 50% of a peptide comprising the amino acid sequence of SEQ IDNO: 3 to a sample from a subject suffering from an infection by Borreliaafzelii. In certain embodiments, the isolated peptide has the amino acidsequence of SEQ ID NO: 3. A sample from a subject suffering frominfection by Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii,and/or Borrelia valaisiana can be evaluated using these compositions inroutine diagnostic methods such as those provided herein. Inembodiments, the subject is suffering from Borrelia afzelii.

The invention provides compositions that include the mixtures of theantigenic components of SEQ ID NO: 3. Such compositions according to theinvention contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc)isolated peptides, wherein the peptides include amino acid sequences ofSEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. The mixtures can includeseparate peptides having the sequences of SEQ ID NO: 7, SEQ ID NO: 8,and SEQ ID NO: 9. The peptides can have sequences of SEQ ID NO: 7, SEQID NO: 8, and SEQ ID NO: 9 joined, for example, by covalent linkage,such as a peptide bond or a peptide linker. A sample from a subjectsuffering from infection by Borrelia burgdorferi, Borrelia afzelii,Borrelia garinii, and/or Borrelia valaisiana can be evaluated usingthese compositions in routine diagnostic methods such as those providedherein. In embodiments, the subject is suffering from Borrelia afzelii.

The invention provides compositions containing an isolated peptidehaving an amino acid sequence at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to SEQ IDNO: 2, wherein a 10-fold molar excess of the peptide inhibits binding ofat least 50% of a peptide comprising the amino acid sequence of SEQ IDNO: 2 to a sample from a subject suffering from an infection by Borreliagarinii. In certain embodiments, the isolated peptide has the amino acidsequence of SEQ ID NO: 2. A sample from a subject suffering frominfection by Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii,and/or Borrelia valaisiana can be evaluated using these compositions inroutine diagnostic methods such as those provided herein. Inembodiments, the subject is suffering from Borrelia garinii.

The invention provides compositions that include the mixtures of theantigenic components of SEQ ID NO: 2. Such compositions according to theinvention contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc)isolated peptides, wherein the peptides include amino acid sequences ofSEQ ID NO: 6 and SEQ ID NO: 9. The mixtures can include separatepeptides having the sequences of SEQ ID NO: band SEQ ID NO: 9. Thepeptides can have sequences of SEQ ID NO: 6 and SEQ ID NO: 9 joined, forexample, by covalent linkage, such as a peptide bond or a peptidelinker. A sample from a subject suffering from infection by Borreliaburgdorferi, Borrelia afzelii, and/or Borrelia garinii can be evaluatedusing these compositions in routine diagnostic methods such as thoseprovided herein. In embodiments, the subject is suffering from Borreliagarinii.

The invention provides compositions that include the mixtures of theantigenic peptide sequences provided herein. Such compositions accordingto the invention contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, etc) isolated peptides, wherein the peptides include amino acidsequences of SEQ ID NO: 1-9. The mixtures can include separate peptideshaving the sequences of SEQ ID NO: 1-9. The peptides can have sequencesof SEQ ID NO: 1-9 joined, for example, by covalent linkage, such as apeptide bond or a peptide linker. A sample from a subject suffering frominfection by Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii,and/or Borrelia valaisiana can be evaluated using these compositions inroutine diagnostic methods such as those provided herein.

The isolated peptides can further be linked to other polypeptidesequences by linkages well-known in the art, including but not limitedto, chemical linkers, peptide bonds, and peptide linkers. For example,peptides of the invention can be linked to one or more of reporterpolypeptide sequences and/or an epitope tag sequences. In certainembodiments, one of the peptides of SEQ ID NO: 1-9 is joined to areporter polypeptide sequence(s) and/or an epitope tag sequence(s). Incertain embodiments, 2, 3, 4, 5, 6, 7, 8, or 9 of peptides having thesequence of SEQ ID NO: 1-9 are joined to a reporter polypeptidesequence(s) and/or an epitope tag sequence(s).

The invention provides nucleic acids encoding one or more of thepeptides (1, 2, 3, 4, 5, 6, 7, 8, or 9) of the invention, alone or in amixture with other nucleic acids encoding one or more polypeptides ofthe invention. In certain embodiments, each of the peptides is encodedby a separate nucleic acid expression construct. In certain embodiments,more than one peptide is encoded by a single nucleic acid expressionconstruct.

The invention provides methods for diagnosing infection by Borrelia sp.in a subject including the steps of:

a) obtaining a sample from a subject,

b) contacting the sample with the peptide composition of any one of theantigenic peptide compositions of the invention, and

c) detecting binding of the peptide to an antibody in the sample,wherein binding is indicative of infection of the subject by Borreliasp.

The invention also provides methods for monitoring therapeutic treatmentresponse in a subject having a Borrelia sp. infection including thesteps of:

a) obtaining a sample from a subject after therapeutic treatment,

b) contacting the sample with the peptide composition of any one of theantigenic peptide compositions of the invention, and

c) detecting binding of the peptide to an antibody in the sample, and

d) correlating binding with the treatment response of the subject,thereby evaluating the therapeutic treatment response of the subject. Inembodiments, the therapeutic treatment involves the administration of animmunogenic composition (e.g., a vaccine).

The invention also provides methods for selecting a treatment regimenfor a subject having a Borrelia sp. infection including the steps of:

a) administering an agent to a subject,

b) obtaining a sample from the subject,

c) contacting the sample with the peptide composition of any one of theantigenic peptide compositions of the invention, and

d) detecting binding of the peptide to an antibody in the sample,wherein decreased binding is indicative that the subject is susceptibleto treatment with the agent, and wherein the treatment regimen comprisesadministering the agent to the subject if the subject is determined tobe susceptible to treatment with the agent.

In the above aspects of the invention, detection of binding of thepeptide and the antibody can be assayed for by any method well-known inthe art. In embodiments, detection includes detection of an enzymaticreaction. Enzymatic reactions can be catalyzed by an enzyme, forexample, luciferase, alkaline phosphatase, or beta-galactosidase. Incertain embodiments, the methods include isolating the antibody-antigencomplex from the sample, for example by binding the antigen-antibodycomplex to a solid substrate. In certain embodiments, theantibody-antigen complex is formed in solution. In embodiments, theassay is an immunoassay. Immunoassays include, but are not limited to,competitive and non-competitive assay systems using techniques such asLIPS, BIAcore analysis, FACS analysis, immunofluorescence,immunocytochemistry, Western blots, radioimmunoassays, ELISA, “sandwich”immunoassays, immunoprecipitation assays, precipitation reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, and protein A immunoassays. See Ausubel et al,eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley &Sons, Inc., New York, which is hereby incorporated by reference.

In the above aspects of the invention, the sample can be any body fluidor tissue from the subject. Samples can be obtained from the subjectusing any method well-known in the art. In embodiments, the sample isblood, plasma, or serum. In embodiments, the sample is donated blood,tissue, or organ.

The invention provides kits including one or more peptides of theinvention. The invention also provides kits including one or morenucleic acids for encoding one or more peptides of the invention. Inembodiments, the kits further include instructions for using thepeptides and/or nucleic acids in any of the methods described herein.

The invention further provides other embodiments that are providedherein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1F include scatter plots showing the antibody titers resultingfrom VlsE-Δ1 (FIG. 1A), DbpB (FIG. 1B), DbpA (FIG. 1C), p39 (BMP) (FIG.1D), Fla (FIG. 1E) and OspC (FIG. 1F) tested by LIPS. The serum samplesincluded 11 EM (erythema migrans), 8 multiple erythema migrans (MEM), 2Lyme palsy, 6 Lyme arthritis, 1 late Lyme neuroborreliosis, post-Lymedisease syndrome (PLDS) patient samples and 8 uninfected controlsamples. Each symbol represents a serum sample from an individualpatient. The geometric mean is shown as the bar. The cut-off value forcalculating sensitivity and specificity is shown by the dotted line andis derived from the mean plus 5 standard deviation (SD) of the 8uninfected controls.

FIG. 2 includes a scatter plot of the antibody responses from the LIPSVOVO test. Shown are results from 11 EM, 8 multiple erythema migrans(MEM), 2 Lyme palsy, 6 Lyme arthritis, 1 late Lyme neuroborreliosis,post-Lyme disease syndrome (PLDS) patient samples and 8 uninfectedcontrol samples. Each symbol represents a serum sample from anindividual patient. The geometric mean is shown as the bar.

FIGS. 3A-3B show that the VOVO antigen has 94% sensitivity and 100%specificity. FIG. 3A includes a scatter plot showing the results from141 serum samples from patients with confirmed Lyme disease, and 59control serum samples. Each symbol represents a serum sample from anindividual patient. Using the cut-off based on the mean plus 5 SD shownby the solid line, the VOVO LIPS test showed 94% sensitivity and 100%specificity. The C6 ELISA on these same samples showed 76% sensitivityand 96% specificity FIG. 3B includes a plot showing the correlationbetween the titer values obtained from LIPS and C6 ELISA. The LIPS titervalues were first log¹⁰ transformed and then analyzed using a Speramanrank correlation. These results showed rs=0.778.

FIGS. 4A-4C include sequences from Borrelia sp. FIG. 4A includessequences showing three exemplary VOVO antigens for Borrelia burgdorferi(Bb, SEQ ID NO: 1), Borrelia garinii (Bg, SEQ ID NO: 2), and Borreliaafzelii (Ba, SEQ ID NO: 3). FIG. 4B includes sequence alignments of VslE(“V” fragments) peptide sequences from Borrelia burgdorferi (Bb SEQ IDNO: 4 and 5), Borrelia garinii (Bg, SEQ ID NO: 6), and Borrelia afzelii(Ba, SEQ ID NO: 7 and 8). Alignments shows sequence homology within thegroup of sequences and pairwise homology between pairs of sequences.FIG. 4C includes a sequence fragment of the OspC (“O” fragment) peptidepresent in Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii(SEQ ID NO: 9). FIG. 4B discloses SEQ ID NOS 4-8, 4-6, 4-5, 7, 4-5 and8,respectived, in order of appearance.

FIG. 5 is a schematic of the pREN2 mammalian expression vector. Featuresindicated are CMV (cytomegalovirus) promoter, the N-terminal FLAGepitope and Ruc. Sequences for Ruc are in bold. cDNAs for tumor antigenswere cloned downstream of Ruc between the BamHI-XhoI sites. Sequences ofthe FLAG-epitope operably linked to luciferase (SEQ ID NO: 10 and 11)and the multiple cloning site (SEQ ID NO: 12 and 13) are provided.(Plasmid is described in Burbelo et al., 2005. BMC Biotechnology. 5:22,which is hereby incorporated by reference).

DEFINITIONS

“Antigenic fragment” and the like are understood as at least thatportion of an antigen capable of being recognized and specifically boundby an antibody present in a subject having or suspected of having aninfection, particularly a Borrelia sp., particularly when the antigenincludes a partial sequence of consecutive amino acids of at least oneof VslE and OspC antigens. When the antigen is a polypeptide, epitopescan be formed both from contiguous amino acids and noncontiguous aminoacids juxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained upon protein denaturing,whereas epitopes formed by tertiary folding are typically lost uponprotein denaturing. Typically, antigenic fragments include at least 3,and more usually, at least 4, 5, 6, 7, 8, or 10 amino acids in a uniquespatial conformation. Moreover, common epitopes for antigens have beenmapped and can be used as antigenic fragments in the compositions andmethods provided herein. Antigenic fragments can include deletions ofthe amino acid sequence from the N-terminus or the C-terminus, or both.For example, an antigenic fragment can have an N- and/or a C-terminaldeletion of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45; 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, or more amino acids. Antigenic fragments can also include one ormore internal deletions of the same exemplary lengths. Antigenicfragments can also include one or more point mutations, particularlyconservative point mutations. In addition, an antigenic fragment (e.g.,a protein) can include the full length, wild-type sequence of theantigen. An antigenic fragment can include more than one potentialantibody binding site.

As used herein, “binding” is understood as having at least a 10² ormore, 10³ or more, preferably 10⁴ or more, preferably 10⁵ or more,preferably 10⁶ or more preference for binding to a specific bindingpartner as compared to a non-specific binding partner (e.g., binding anantigen to a sample known to contain the cognate antibody). That anantibody “specifically binds” to an antigen, epitope or protein meansthat the antibody reacts or associates more frequently, more rapidly,with greater duration, with greater affinity, or with some combinationof the above to an antigen, epitope or protein than with alternativesubstances, including unrelated proteins.

As used herein, “Borrelia sp.” is understood as any Borrelia speciesknown to cause Lyme disease, for example, Borrelia burgdorferi (Bb),Borrelia afzelii, Borrelia garinii, and Borrelia valaisiana.

As used herein, “changed as compared to a control” sample or subject isunderstood as having a level of the analyte or diagnostic or therapeuticindicator to be detected at a level that is statistically different thana sample from a normal, untreated, or control sample. Control samplesinclude, for example, cells in culture, one or more laboratory testanimals, or one or more human subjects. Methods to select and testcontrol samples are within the ability of those in the art. An analytecan be a naturally occurring substance that is characteristicallyexpressed or produced by the cell or organism (e.g., an antibody, aprotein) or a substance produced by a reporter construct (e.g,β-galactosidase or luciferase). Depending on the method used fordetection the amount and measurement of the change can vary. Changed ascompared to a control reference sample can also include a change in oneor more signs or symptoms associated with or diagnostic of Borrelia sp.infection. Determination of statistical significance is within theability of those skilled in the art, e.g., the number of standarddeviations from the mean that constitute a positive result.

As used herein, the terms “comprises,” “comprising,” “containing,”“having” and the like can have the meaning ascribed to them in U.S.Patent law and can mean “includes,” “including,” and the like;“consisting essentially of” or “consists essentially” likewise has themeaning ascribed in U.S. Patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic ornovel characteristics of that which is recited is not changed by thepresence of more than that which is recited, but excludes prior artembodiments. As used herein in reference to antigenic polypeptides,“consisting essentially of” is understood as an antigenic polypeptidesequence including the claimed sequence, and optionally furthercontaining other elements or optionally having shorter amino acidsequences than presented that do not materially affect the basic andnovel characteristics of the antigenic polypeptide. That is, otherelements or deletion of sequences that neither substantially inhibit orenhance binding of the peptide to cognate antibodies in a subjectsample, or decrease the specificity of the binding of the antigen to asubject sample. In certain embodiments, antigenic fragments of longerpolypeptides can be expressed to include an initiator methionine, asignal sequence for translocation of the protein, or may includesequences at the N- or C-terminus after cleavage with a protease notpresent in the native sequence. As used herein, a polypeptide consistingessentially of an antigenic fragment can be linked covalently (e.g., bya peptide bond or other linkage) to a second polypeptide, for example areporter polypeptide, or an epitope tag (e.g., a FLAG tag). In anantigen with multiple domains or binding sites, the antigenic domainscan be covalently linked by any type of linkage that does not disruptbinding to the antigenic site, e.g., through any of a number of chemicalcovalent linkages (e.g., cross-linking reagents commercially availablefrom Pierce, a part of Thermo Fisher Scientific, and other sources), orthrough peptide bonds, including peptide sequences typically referred toas linker sequences or peptide linkers.

“Contiguous” is understood as touching or connected to through anunbroken sequence.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “diagnosing” and the like as used herein refers to a clinical orother assessment of the condition of a subject based on observation,testing, or circumstances for identifying a subject having a disease,disorder, or condition based on the presence of at least one indicator,such as a sign or symptom of the disease, disorder, or condition.Typically, diagnosing using the method of the invention includes theobservation of the subject for multiple indicators of the disease,disorder, or condition in conjunction with the methods provided herein.A diagnostic methods provide an indicator that a disease is or is notpresent. A single diagnostic test typically may not provide a definitiveconclusion regarding the disease state of the subject being tested.

As used herein, “epitope tag” is understood as a peptide sequenceunrelated to the peptide to which it is attached that provides aspecific antigen (e.g., myc tag, HAI tag, FLAG tag) or binding site(e.g., GST, 6×His (SEQ ID NO: 14)) for which a commercially availablereagent (e.g., monoclonal antibody, affinity matrix) is available tofacilitate isolation of a molecule including the epitope tag.

As used herein, the terms “identity” or “percent identity”, refers tothe subunit sequence similarity between two polymeric molecules, e.g.,two polynucleotides or two polypeptides. When a subunit position in bothof the two molecules is occupied by the same monomeric subunit, e.g., ifa position in each of two peptides is occupied by serine, then they areidentical at that position. The identity between two sequences is adirect function of the number of matching or identical positions, e.g.,if half (e.g., 5 positions in a polymer 10 subunits in length), of thepositions in two peptide or compound sequences are identical, then thetwo sequences are 50% identical; if 90% of the positions, e.g., 9 of 10are matched, the two sequences share 90% sequence identity. The identitybetween two sequences is a direct function of the number of matching oridentical positions. Thus, if a portion of the reference sequence isdeleted in a particular peptide, that deleted section is not counted forpurposes of calculating sequence identity. Identity is often measuredusing sequence analysis software e.g., BLASTN or BLASTP (available atwww.ncbi.nih.gov/BLAST). Additional, computer programs for determiningidentity are well-known in the art.

As used herein, “immunoassay” includes any of a number of antibody basedassays including LIPS, ELISA, RIA, immunoprecipitation assay, dot blot,slot blot, immunofluorescence, and immunohistochemistry. In certainembodiments, immunoassay does not include western blots.

As used herein, “isolated” or “purified” when used in reference to apolypeptide or, nucleic acid means that a naturally occurringpolypeptide or nucleic acid has been removed from its normalphysiological environment (e.g., protein isolated from plasma or tissue,optionally bound to another protein) or is synthesized in a non-naturalenvironment (e.g., artificially synthesized in an in vitro transcriptionor translation system or using chemical synthesis, fragments amplifiedby PCR and/or generated by restriction digest). Thus, an “isolated” or“purified” polypeptide can be in a cell-free solution or placed in adifferent cellular environment (e.g., expressed in a heterologous celltype). The term “purified” does not imply that the polypeptide is theonly polypeptide present, but that it is essentially free (about 90-95%,up to 99-100% pure) of cellular or organismal material naturallyassociated with it, and thus is distinguished from naturally occurringpolypeptide. Similarly, an isolated nucleic acid is removed from itsnormal physiological environment. “Isolated” when used in reference to acell means the cell is in culture (i.e., not in an animal), either cellculture or organ culture, of a primary cell or cell line. Cells can beisolated from a normal animal, a transgenic animal, an animal havingspontaneously occurring genetic changes, and/or an animal having agenetic and/or induced disease or condition. An isolated virus or viralvector is a virus that is removed from the cells, typically in culture,in which the virus was produced.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

As used herein, “kits” are understood to contain at least onenon-standard laboratory reagent for use in the methods of the inventionin appropriate packaging, optionally containing instructions for use.The kit can further include any other components required to practicethe method of the invention, as dry powders, concentrated solutions, orready to use solutions. In some embodiments, the kit comprises one ormore containers that contain reagents for use in the methods of theinvention; such containers can be boxes, ampules, bottles, vials, tubes,bags, pouches, blister-packs, or other suitable container forms known inthe art. Such containers can be made of plastic, glass, laminated paper,metal foil, or other materials suitable for holding reagents.

As used herein, a “nucleic acid encoding a polypeptide” is understood asany possible nucleic acid that upon (transcription and) translationwould result in a polypeptide of the desired sequence. The degeneracy ofthe nucleic acid code is well understood. Further, it is well-known thatvarious organisms have preferred codon usage, etc. Determination of anucleic acid sequence to encode any polypeptide is well within theability of those of skill in the art.

As used herein, “operably linked” is understood as joined, preferably bya covalent linkage, e.g., joining an amino-terminus of one peptide;expressing an enzyme to a carboxy terminus of another peptide;expressing a signal sequence to target the protein to a specificcellular compartment; or joining a promoter sequence with a proteincoding sequence, in a manner that the two or more components that areoperably linked either retain their original activity, or gain anactivity upon joining such that the activity of the operably linkedportions can be assayed and have detectable activity, e.g., enzymaticactivity or protein expression activity.

As used herein, “plurality” is understood to mean more than one. Forexample, a plurality refers to at least two, three, four, five, or more.

As used herein, “peptide linker” and the like are understood to be aminoacid sequences of essentially any length (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids) and arerelatively non-antigenic. Peptide linker sequences can be encoded bynucleic acid sequences between nucleic acid sequences encodingfunctional components of the peptide, e.g., antigenic peptide sequences,reporter peptide sequences, epitope tag sequences, etc. Alternatively,peptide linkers can be short peptide sequences including reactivegroups, typically at the termini of the peptide that can be used to jointhe linker sequences to peptides to allow them to be covalently linked.

A “polypeptide” or “peptide” as used herein is understood as two or moreindependently selected natural or non-natural amino acids joined by apeptide bond. A peptide can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more natural or non-natural aminoacids joined by peptide bonds. Polypeptides as described herein includefull length proteins (e.g., fully processed proteins), shorter aminoacids sequences (e.g., fragments of naturally occurring proteins orsynthetic polypeptide fragments), or artificial peptide sequencescomposed of naturally occurring and/or non-naturally occurring peptidesequences.

As used herein, a “reporter protein” or a “reporter polypeptide” isunderstood as a polypeptide that can be readily detected, preferablyquantitatively detected, either directly or indirectly. A reporterpolypeptide typically has an enzymatic activity, luciferase activity,alkaline phosphatase activity, beta-galactosidase activity, acetyltransferase activity, or the like, wherein catalysis of a reaction witha substrate by the enzyme results in the production of a product, e.g.,light, that can be detected at a specific wavelength of light,radioactivity, or the like, such that the amount of the reporter peptidecan be determined in the sample, either as a relative amount, or as anabsolute amount by comparison to control samples.

A “sample” as used herein refers to a biological material that isisolated from its environment (e.g., blood or tissue from an animal,cells, or conditioned media from tissue culture) and is suspected ofcontaining, or known to contain an analyte, such as an antibody. Asample can also be a partially purified fraction of a tissue or bodilyfluid (e.g., serum or plasma). A reference sample or a control samplecan be a sample from a donor not having the disease or condition,including fluid or tissue from a subject. A reference or control samplecan also be from an untreated donor or cell culture not treated with anactive agent (e.g., no treatment or administration of vehicle only). Areference or control sample can also be taken at a time point prior tocontacting the cell or subject with an agent or therapeutic interventionto be tested or at the start of a prospective study.

“Sensitivity and specificity” are statistical measures of theperformance of a binary classification test. The sensitivity (alsocalled recall rate in some fields) measures the proportion of actualpositives which are correctly identified as such (e.g., the percentageof sick people who are identified as having the condition); and thespecificity measures the proportion of negatives which are correctlyidentified (e.g., the percentage of well people who are identified asnot having the condition). They are closely related to the concepts oftype I and type II errors. A theoretical, optimal prediction can achieve100% sensitivity (i.e., predict all people from the sick group as sick)and 100% specificity (i.e., not predict anyone from the healthy group assick).

The concepts are expressed mathematically as follows:sensitivity=# true positives/# true positives+# false negativesspecificity=# true negatives/# true negatives+# false positives.

A “subject” as used herein refers, to an organism. In certainembodiments, the organism is an animal. In certain embodiments, thesubject is a living organism. In certain embodiments, the subject is acadaver organism. In certain preferred embodiments, the subject is amammal. In certain embodiments, the subject is a domesticated mammal ora primate including a non-human primate. In certain preferredembodiments, the subject is a mammal that is capable of being infectedby a sp. Borrelia pathogen. Examples of subjects include humans;monkeys, dogs, cats, mice, rats, cows, horses, goats, and sheep. A humansubject may also be referred to as a patient.

A “subject sample” can be a sample obtained from any subject, typicallya blood or serum sample, however the method contemplates the use of anybody fluid or tissue from a subject. The sample may be obtained, forexample, for diagnosis of a specific individual for the presence orabsence of sp. Borrelia pathogen infection. In certain embodiments, asubject sample can be a sample for screening of a subject tissue(solubilized or treated to release antibodies) or body fluid (e.g.,blood, serum, plasma) prior to transplant or transfusion into arecipient.

A subject “suffering from,” “suspected of suffering from,” or “having” aspecific disease, condition, or syndrome has a sufficient number of riskfactors or presents with a sufficient number or combination of signs orsymptoms of the disease, condition, or syndrome such that a competentindividual would diagnose or suspect that the subject has the disease,condition, or syndrome. Methods for identification of subjects sufferingfrom or suspected of suffering from conditions such as sp. Borreliapathogen infection is within the ability of those in the art. Subjectssuffering from, and suspected of suffering from, a specific disease,condition, or syndrome are not necessarily two distinct groups.

As used herein, “susceptible to” or “prone to” or “predisposed to” aspecific disease or condition and the like refers to an individual whobased on genetic, environmental, health, and/or other risk factors ismore likely to develop a disease or condition than the generalpopulation. An increase in likelihood of developing a disease may be anincrease of about 10%, 20%, 50%, 100%, 150%, 200%, or more.

As used herein, a “VOVO antigen” is understood as an antigen thatincludes isolated peptides having sequences of at least one of SEQ IDNO: 4-8 and SEQ ID NO: 9. The peptides can be separate peptides. Thepeptides can be linked directly by a covalent linkage such as a peptidebond. The peptides can be linked by a peptide linker sequence. Peptidesequences of SEQ ID NO: 4-9 are identical to or significantly identicalto portions of the VlsE C6 and OspC peptides of a Borrelia sp. bacteria,particularly one of Borrelia burgdorferi (Bb), Borrelia garinii (Bg),Borrelia afzelii (Ba), and Borrelia valaisiana. The VOVO antigensprovided herein are based on protein sequences of one of the Borreliasp. However, VOVO antigens can include sequences from more than oneBorrelia sp. An exemplary Borrelia burgdorferi VOVO antigen compositionincludes at least the peptide sequences amino acids 1-26 of SEQ ID NO: 1(i.e., SEQ ID NO: 4), a fragment corresponding to the VlsE-Δ1 protein;amino acids 30-40 of SEQ ID NO: 1 (i.e., SEQ ID NO: 5), a fragmentcorresponding to the OspC protein; and amino acids 41-65 of SEQ ID NO: 1(i.e., SEQ ID NO: 9), a fragment corresponding to the VlsE-Δ2 protein;and/or a composition that has a peptide sequence that is at least 80%,optionally at least 85% identical, at least 90% identical, or at least95% identical to SEQ ID NO: 1, wherein a 10-fold molar excess of thepeptide or a combination of the peptide sequences, inhibits binding ofat least 50% of a peptide comprising the amino acid sequence of SEQ IDNO: 1 to a sample from a subject suffering from an infection by aBorrelia burgdorferi pathogen. Competition assays can be performed usingany method known in the art, for example, using a BIACORE device.

Similarly, an exemplary Borrelia garinii (Bg) VOVO antigen can includeisolated peptide sequences SEQ ID NO: 6 and SEQ ID NO: 9; and/or acomposition that has a peptide sequence that is at least 80%, optionallyat least 85% identical, at least 90% identical, or at least 95%identical to SEQ ID NO: 2, wherein a 10-fold molar excess of the peptideor a combination of the peptide sequences, inhibits binding of at least50% of a peptide comprising the amino acid sequence of SEQ ID NO: 2 to asample from a subject suffering from an infection by a Borrelia gariniipathogen.

Similarly, an exemplary Borrelia afzelii (Ba) VOVO antigen can includeisolated peptide sequences SEQ ID NO: 7 or 8 and SEQ ID NO: 9; and/or acomposition that has a peptide sequence that is at least 80%, optionallyat least 85% identical, at least 90% identical, or at least 95%identical to SEQ ID NO: 3, wherein a 10-fold molar excess of the peptideor a combination of the peptide sequences, inhibits binding of at least50% of a peptide comprising the amino acid sequence of SEQ ID NO: 3 to asample from a subject suffering from an infection by a Borrelia afzeliipathogen.

A VOVO antigen can also be generated for detection of Borrelia sp.infection from any combination of Borrelia burgdorferi, Borreliaafzelii, Borrelia garinii, and Borrelia valaisiana for the detection ofLyme disease. The VOVO antigen can be a mixed VOVO antigen including amixture of any combination of peptides having at least 80% sequenceidentity to each of SEQ ID NO: 1, 2, or 3, wherein a 10-fold molarexcess of the peptide inhibits binding of at least 50% of a peptidecomprising the corresponding amino acid sequence of SEQ ID NO: 1, 2, or3 to a sample from a subject suffering from an infection by thecorresponding species of Borrelia. In certain embodiments, a genericBorrelia sp. VOVO antigen can be prepared using a combination ofisolated peptide sequences of at least one of SEQ ID NO: 4 or 5; SEQ IDNO: 6; at least one of SEQ ID NO: 7 or 8; and SEQ ID NO: 9, wherein a10-fold molar excess of the peptide or a combination of the threepeptide sequences, inhibits binding of at least 50% of a peptidecomprising the amino acid sequence of SEQ ID NO: 1, 2, or 3 to a samplefrom a subject suffering from an infection by a Borrelia burgdorferi(Bb) pathogen, Borrelia garinii (Bg) pathogen, or a Borrelia afzelii(Ba) pathogen, respectively.

The VOVO antigen can be a mixture of peptides, optionally wherein thepeptides are covalently linked to each other (e.g., peptide bond). Incertain embodiments, the mixed VOVO antigen is expressed from a singlenucleic acid. A mixed VOVO antigen need not be designed for use in thediagnosis of all Borrelia sp. infections. In certain embodiments, themixed VOVO antigen can be an antigen for detection of both Borrelia sp.present in Eurasia (Borrelia afzelii and Borrelia garinii). A subjectwill typically know if a potential Lyme infection was picked up in theUS or Eurasia. However, a single test to detect antibodies to any of thespecies of Borrelia known to cause Lyme disease could allow for the useof a single test internationally.

The fragments corresponding to each of the VlsE and OspC peptides can bemixed together and used in an immunoassay. When the peptides areexpressed separately, preferred assay methods include those in which theantigen is bound to a solid surface prior to contacting the antigen witha serum sample (e.g., ELISA assay). The combination of the sequences inclose proximity to each other allows for the binding of the polyclonalantibodies produced in an immune response to bind to more than one siteon the surface on which the antigens are coated, increasing the apparentaffinity of the antibodies. When binding of the antigens to theantibodies present in serum is performed in solution (e.g., in animmunoprecipitation assay such as a LIPS assay), it is preferred thatthe peptide sequences are covalently linked to each other. A peptideincluding all of the fragments can be easily produced using anexpression construct in which the three sequences corresponding to afragment of each of the VlsE-Δ1, VlsE-Δ2, and OspC from the desiredBorrelia sp. joined in frame, optionally by linker sequences, andoperably linked to a promoter sequence appropriate for the system inwhich the protein is to be expressed. Although the VOVO peptideexemplified in the instant application, includes domains in thefollowing order: VlsE-Δ1, OspC, VlsE-Δ2, and OspC, it is understood thatany combination or order of the fragments of the protein sequences ispossible, e.g., VlsE-Δ1-VlsE-Δ21-OspC; VlsE-Δ1-OspC-VlsE-Δ2;OspC-VlsE-Δ1-VlsE-Δ2; VlsE-Δ2-VlsE-Δ1-OspC; VlsE-Δ2-OspC-VlsE-Δ1;OspC-VlsE-Δ2-VlsE-Δ1; etc. Analogous combinations can be made based onthe sequences SEQ ID NO: 6 and 9. Analogous combinations can be madebased on the sequences SEQ ID NO: 7, 8, and 9. It is understood thatmore than three fragments can be joined together (e.g., 4, 5, 6, 7, 8,9, 10, etc.), e.g., to facilitate expression of the peptide sequences,provide desired stoichiometry and/or relative positions of thefragments, etc. It is understood that VlsE-Δ1, VslE Δ2, and OspCfragments from different Borrelia sp. can be mixed or joined together.It is further understood that VOVO antigens can be linked to otherprotein sequences, e.g., reporter constructs, epitope tags, etc.

In an alternative embodiment, the “V” and/or “O” fragments can beexpressed with an epitope tag, e.g., a 6×His tag (SEQ ID NO:14), andcoated onto a bead for binding to antibodies present in sera.

In an alternative embodiment, the “V” and/or “O” fragments can be joinedusing any of a number of commercially available cross-linking reagents.The fragments can be joined in any order or any orientation as long asthe linking agents do not disrupt binding of the antibodies to theantigen.

Other methods for joining the “V” and/or “O” fragments are well-known tothose of skill in the art.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. This includes all individual sequences when arange of SEQ ID NOs is provided. For example, a range of 1 to 50 isunderstood to include any number, combination of numbers, or sub-rangefrom the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

DETAILED DESCRIPTION OF THE INVENTION

Currently, there is a need for sensitive and specific testing toidentify and monitor Lyme-infected individuals. A variety ofimmunoassays, including immunofluorescence assays, Western blot, andELISAs have been employed to detect antibodies to Bb proteins. One ofthe most useful approaches employs defined short peptides derived fromVslE and OspC for testing (Bacon, et al., 2003. J Infect Dis187:1187-99; Embers, et al., 2007. Clin Vaccine Immunol 14:931-6.;Liang, et al., 1999. J Immunol 163:5566-73; and Liang, et al., 1999. JClin Microbiol 37:3990-6). For example, the most sensitive ELISA usingthe C6 peptide of VslE, matches the 2-tiered Western blotting insensitivity and specificity. While there is an interest in using the C6ELISA and other serological tests for monitoring antibiotic therapy ofLyme infected patients, these studies are hampered by the limiteddynamic range of these solid phase immunoassays and the need for timeconsuming and cumbersome serum dilutions to obtain values in the linearrange (Fleming, et al., 2004. Eur J Clin Microbiol Infect Dis 23:615-8;Levy, et al., 2008. Clin Vaccine Immunol 15:115-9; Philipp, et al.,2003. J Clin Microbiol 41:4955-60; and Philipp, et al., 2005. Clin DiagnLab Immunol 12:1069-74).

The serological laboratory tests most widely available and employed arethe Western blot and ELISA. A two-tiered protocol is recommended by theCDC: the sensitive ELISA test is performed first, and if it is positiveor equivocal then the more specific Western blot is run (Wilske et al.2005. Ann. Med. 37: 568-79). However, Western blots are cumbersome,rarely used in clinical laboratories making the tests more inconvenient,and are not adaptable to high throughput methods. The reliability oftesting in diagnosis remains controversial, however, studies show thatthe IgM Western blot has a specificity of 94-96% for patients withclinical symptoms of early Lyme disease. The initial ELISA test has asensitivity of about 70%, and in two tiered testing, the overallsensitivity is only 64% although this rises to 100% in a subset ofpeople with disseminated symptoms, such as arthritis.

Luciferase Immunoprecipitation System (LIPS) is a highly sensitiveimmunoprecipitation technology that utilizes mammalian cell-produced,recombinant fusion protein antigens for efficiently evaluating antibodyresponses (see US Patent Publication 2007/0259336 and Burbelo et al.2005. BMC Biotechnol. 5:22, which are hereby incorporated by reference).LIPS shows strong diagnostic performance for detecting antibodies toinfectious agents (e.g., HCV, HIV, HTLV-I, and filarial infectiousagents) and provides new tools to monitor drug treatment andsub-stratify disease states. LIPS is highly useful for profilingautoimmunity and in one study showed several advantages over a highlysensitive radioactive in vitro transcription/translation assay fordetecting anti-IA2 autoantibodies associated with type I diabetes.

LIPS is based on fusing protein antigens to a light emitting enzymereporter, Renilla luciferase (Ruc), and then using these antigens inimmunoprecipitation assays with sera samples and Protein A/G beads.Following washing, light production is measured yielding highlyquantitative antibody titers. While LIPS has already shown highsensitivity for detecting fungal, helminthic, filarial, and a variety ofviral infection agents, its utility to accurately evaluate humoralresponses to bacterial pathogen antigens has not been assessed. Asdescribed below, LIPS was used to evaluate antibody responses to a panelof Bb antigens for the serological diagnosis of Lyme disease. Followingthe evaluation of the training set, several antigens including VslE,DbpA, Dbp-B, BMP, and FlaB, showed high performance. Nevertheless, theantigen with the greatest dynamic range of detection and highestsensitivity and specificity was composed of a synthetic gene (designatedVOVO) containing 2 alternating copies of immunoreactive peptides derivedfrom VslE and OspC antigens. Analysis of an independent validation serumset with VOVO showed 94% sensitivity and 100% specificity, and markedlyout-performed the C6 ELISA. Without serum dilution, LIPS also showed awide dynamic range of detection making it a practical tool foraccurately quantifying anti-Lyme antibodies. These results demonstratethat an immunoassay based on the VOVO antigen, preferably LIPS screeningwith VOVO antigen, optionally with other Borrelia sp. proteins (e.g.,VslE-Δ1 (Accession No: GU182319), VslE-Δ2 (Accession No: GU182320); Fla(GenBank AAC66541); Bmp (GenBank NP_(—)212517); Dbp (GenBank AAC70025),DBpA (GenBank YP_(—)002455347), OspC2-Δ1 (GenBank NP_(—)047005)) orfragments therefrom, is an efficient high-throughput method foraccurately determining anti-Lyme patient antibody responses, includingwithout serum dilution.

The test can be used as part of a routine screening panel for sp.Borrelia pathogen progression or regression in a subject by detectingbinding of antibodies to VOVO antigens.

Further, the method can be used for monitoring donated blood, organs,and/or tissues for the presence of sp. Borrelia pathogen infection.

EXAMPLES

It should be appreciated that the invention should not be construed tobe limited to the examples that are now described; rather, the inventionshould be construed to include any and all applications provided hereinand all equivalent variations within the skill of the ordinary artisan.

Example 1 Material and Methods

Patient Sera. Serum was obtained from patients or volunteers underinstitutional review board approved protocols at the Clinical Center,National Institute of Allergy and Infectious Diseases, NIH. The initialtraining set (n=46) included serum from 11 EM, 8 multiple erythemamigrans (MEM), 2 Lyme palsy, 6 Lyme arthritis, 1 late Lymeneuroborreliosis, 10 post-Lyme disease syndrome (PLDS) subject samples,and 8 uninfected control sera subject samples were analyzed. Testing ofthe validation set consisted of 225 coded serum samples. This validationcohort consisted of 59 control sera and 141 samples from patients withestablished Lyme disease. The codes for the validation cohort was brokenonly after titers were established and categorization of Lyme infectionstatus had been made. The antibody titer results for the validationcohort obtained by LIPS was also compared with the C6 ELISA. Of note, anadditional 40 samples with uncertain diagnosis for Lyme disease werealso analyzed by LIPS and the C6 ELISA, but were not used in calculationof sensitivity and specificity.

Generation of Ruc-Antigen Fusion Constructs. pREN2, a mammalian Renillaluciferase (Ruc) expression vector, was used to generate all plasmids(FIG. 3). Bb genes were amplified by PCR specific linker-primer adaptersusing synthetic cDNA templates assembled in the investigators laboratoryor obtained from Blue Heron Biotechnology (Seattle, Wash.).Gene-specific primers were then used in PCR amplifications forgenerating cDNA sequences for cloning as C-terminal fusions of Ruc. Foreach C-terminal fusion, a stop codon was included at the end of thecoding sequence. The nucleotide and protein sequence for VOVO hasGenBank Accession number GU134803. The peptide encoded by VOVO isMKKDDQIAAAIALRGMAKDGKFAVKELTSPVVAESPKKPMKKDDQIAAAMVLRGMAKDGOQFALKPVVAESPKKP (SEQ ID NO: 15), in which thepeptide sequence from VslE is underlined and the peptide sequence fromOspC is in italics.Two constructs, VslE-Δ1 and VslE-Δ2, containing theVslE peptide sequences have GenBank Accession sequences GU182319 andGU182320 were also tested. DNA sequencing was used to confirm theintegrity of all the DNA constructs. Plasmid DNA was then prepared fromthe different pREN2 expression vectors using a Qiagen Midi preparationkit.

LIPS Analysis. Following transfection of mammalian expression vectors,crude protein extracts were obtained as described in Burbelo, et al.2008. Biochem Biophys Res Commun 366:1-7, which is hereby incorporatedby reference.). A detailed protocol of the LIPS assay is now availablealong with a corresponding technical video from the Journal ofVisualized Experiments (Burbelo et al., 2009, J. Vis. Exp.www.jove.com/index/Details.stp?ID=1549).

Data Analysis. The GraphPad Prism software (San Diego, Calif.) was usedfor statistical analysis. Results for qualitative antibody titersbetween the controls and Bb-infected individuals are reported as themean+5 standard deviation (SD). Mann-Whitney U tests were used tocompare the antibody titers among the groups.

Example 2 LIPS Detection of Antibody Responses to a Panel of Bb Antigens

Previous studies from various laboratories have identified a largenumber of Bb antigens useful for serological screening of Lyme disease.Fifteen different Bb antigens including FlaB, BMP, Dbp-A, DbpB, OspC,OspA, Bbk and 2 different VlsE constructs were initially syntheticallyassembled and constructed as C-terminal fusion with Ruc. LIPS evaluationof these different antigens began by testing a small cohort of serumsamples (n=44) consisting of serum from 11 EM, 8 MEM, 2 Lyme palsy, 6Lyme arthritis, 1 late Lyme neuroborreliosis, 10 PLDS subjects, and 8uninfected control subject serum samples. To easily visualize thediffering immunoreactivity to this large antigen panel, we employed ourpreviously described heat map analysis to graphically display theantibody responses using a login scale to the most informative antigens(FIG. 1A). From these tests, 6 of these Bb proteins showed weak ornon-existent antibody signals (BBk, OspA, OspF, Crasp, OspC, DbpA (withsignal peptide), while 7 others (VlsE-Δ1, VlsE-Δ2; Fla; Bmp; DbpAwithout signal peptide, DbpB without signal peptide, OspC2-Δ1 withoutsignal peptide) showed high levels of immunoreactivity with over 50% ofthe Lyme samples (FIG. 1A). Based on the mean plus 5 standarddeviations, the most informative antigen in the initial panel wasVlsE-Δ1 and was followed by VslE-Δ2. Other antigens such as DbpA andDbp-B showed nearly identical serological activity but were lesssensitive.

Next, a new synthetic antigen was generated and tested. This new antigenwas synthetically assembled in one recombinant protein. The new antigenwas designated VOVO and contained 2 alternating copies of immunoreactivepeptides derived from VslE and OspC antigens. The rational behind VOVO'sdesign was that the repeated antigenic peptides from the 2 differentproteins might increase the valency, and thereby apparent affinity, andcapture low affinity antibodies. From LIPS testing, VOVO appeared to bean effective antigen. Without wishing to be bound by mechanism, it issuggested that the use of the fragments provided herein allows for theunmasking of hidden or cryptic immunodominant antigen sequences to allowfor binding of serum antibodies. The mean anti-VOVO antibody titer inthe 38 Lyme samples was 1,716,000 LU and was over 1000-fold higher thanantibody titer of 1,395 LU in the controls (Mann Whitney U test,P<0.0017). Using a cut-off derived from the mean plus 3 SD of thecontrols, 84% of the Lyme samples were VOVO positive and all the serafrom uninfected controls were negative (FIG. 1B), Only a few serumsamples including 1 PLDS and 4 EM samples were seronegative foranti-VOVO antibodies, which were also negative by C6 ELISA. Thesepromising results indicate that VOVO is a highly useful antigen for LIPSscreening of Lyme sera, including in the early stage after infection,e.g., within 1-2 weeks of infection.

Example 3 Using VOVO with a New Independent Validation Cohort

To test the effectiveness of VOVO and compare it with the C6 ELISA, anew validation cohort of 225 blinded sera were evaluated. Followingbreaking the code, the LIPS antibody titer data were analyzed. Similarto the training set, the mean anti-VOVO antibody titer in the 141 Lymesamples was 589,200 LU and was markedly higher than antibody titer of537 LU in the 59 controls (Mann Whitney U test, P<0.0017). In order todetermine the sensitivity and specificity, a diagnostic cut-off 5 valueof LU based on the mean plus 5 SD of the control samples was used. Usingthis cut-off, the VOVO LIPS test showed 94% sensitivity and 100%specificity with these samples (FIG. 2A). The C6 ELISA had a markedlylower diagnostic performance of 76% sensitivity and 98% specificity. ROCanalysis showed that the area under the curves for antibodies to LIPSand C6 ELISA were statistically different (p=0.005). The VOVO LIPS testsalso had a markedly greater dynamic range of detection (FIG. 2A) and didnot require dilution. However, as shown in FIG. 2B, correlation of log¹⁰transformed LIPS values with the C6 ELISA values showed that both assaystracked each other (rs=0.82, p<0.00001).

Example 4 Generation of a VOVO Antigen for Detection of Antibodies toOther Borrelia sp

Based on the analysis performed for antigens of Bb, antigens wereselected based on homology with the VslE sequences selected above fromother Borrelia sp. Alignments of the Bb VslE-Δ1 and VslE-Δ2 antigensequences against sequences from VslE proteins from one B. garinii andtwo B. afzelii sequences are shown in FIG. 4B. The OspC fragmentsequence in the Bb VOVO antigen has the same sequence as is found in B.garinii and two B. afzelii. As such, these sequence alignments indicatethat B. garinii and B. afzelii can be useful in the context of a VOVOantigen for detection of infection with a pathogen from those species.

Upon generation of VOVO antigens for B. garinii and B. afzelii, theantigens are validated using the method provided in the example above. Aset of samples from subjects known to have been infected with either B.garinii or B. afzelii are analyzed for binding to the VOVO antigen. Thesamples are then unblinded the sensitivity and specificity of the testare analyzed. In addition, an assay to detect infection by B. garinii orB. afzelii using the VOVO antigen containing Bb sequences can be testedto determine if there is sufficient cross-reactivity to allow theantigens to be used cross-species.

Alternatively, other proteins from B. garinii and B. afzelii are testedfor their utility as diagnostic antigens. For example, full lengthproteins and fragments of B. garinii and B. afzelii proteins FlaB, BMP,Dbp-A, DbpB, OspC, OspA, Bbk, and VslE are tested for their utility asantigens for detection of infection with B. garinii or B. afzelii.

All references, patents, patent applications, and GenBank numbers as ofthe date of filing of the instant application are hereby incorporated byreference as if they were each incorporated individually.

Borrelia afzelii - OspCMKKNTLTAILMTLFLFISCNNSGKVGILTSTNPADESAKGPNLTEISKKITDSNAFVLAVKEVETLVLSIDELAKKAIGQKIDNNNGLAALNNQNGSLLAGAYAISTLITEKLSKLKNLEELKTEIAKAKKCSEEFTNKLKSGHADLGKQDATDDHAKAAILKTHATTDKGAKEFKDLFESVEGLLKAAQVALTNSVKELTSPVVAESPKKP* (SEQ ID NO: 16)Borrelia burgdorferi - OspCMKKNTLSAILMTLFLFISCNNSGKDGNTSANSADESVKGPNLTEISKKITDSNAVLLAVLEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAISTLIKQKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGTKTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKP* (SEQ ID NO: 17)Borrelia garinii - OspCmkkntlsail mtlflfiscn nsggdtastn pdesakgpnl tviskkitds nafvlavkevealissidel ankaigkvih qnnglnanag qngsllagay aistlitekl sklknseelnkkieeaknhs eaftnrlkgs haqlgvaaat ddhakeailk snptkdkgak elkdlsesveslakaacqeal ansvkeltnp vvaespkkp (SEQ ID NO: 18) FlaB (flagellin; p41)Amino acids 20-336 of B.B. B31 strain (GenBank AAC66541)                       20 anlsktqekl ssgyrinras ddaagmgvsg kinaqirgls 61 qasrntskai nfiqttegnl nevekvlvrm kelavqsgng tysdadrgsi qieieqltde121 inriadqaqy nqmhmlsnks asqnvrtaee lgmqpakint paslsgsqas wtlrvhvgan181 qdeaiavniy aanvanlfsg egaqtaqaap vqegvqqega qqpapataps qggvnspvnv241 tttvdantsl akienairmi sdqranlgaf qnrlesikns teyaienlka syaqikdatm301 tdevvaattn siltqsamam iaqanqvpqy vlsllr*336 (SEQ ID NO: 19) BmpAAmino acids 26-339 of B.b. B31 strain (GenBank NP_212517 )                            26 seipk vsliidgtfd dksfnesaln gvkkvkeefk 61 ielvlkesss nsylsdlegl kdagsdliwl igyrfsdvak vaalqnpdmk yaiidpiysn121 dpipanlvgm tfraqegafl tgyiaaklsk tgkigflggi egeivdafry gyeagakyan181 kdikistqyi gsfadleagr svatrmysde idiihhaagl ggigaievak elgsghyiig241 vdedqaylap dnvitsttkd vgralnifts nhlktntfeg gklinyglke gvvgfvrnpk301 misfelekei dnlsskiink eiivpsnkes yekflkefi*339 (SEQ ID NO: 20)DbpB amino 25-280 of B.b. sensu lato (GenBank AAC70025)                           25 alesss kdlknkilki kkdatgkgvl feaftglktg 61 skvtsgglal reakvqaive tgkflkiiee ealklketgn sgqflamfdl mlevvesled121 vgiiglkarv leesknnpin taerllaaka qienqlkvvk ekqniengge kknnkskkkk*(SEQ ID NO: 21)DbpA amino 33-194 of B.b. of Zs7 strain (GenBank YP_002455347)                                    33 etkiiler sakdiidein kikkdaadnn 61 vnfaafkedk tgskvsensf ileakmrgtt vaekfvtaie geatklkktg ssgefsamyn121 mmlevsgple elgvlrmtkt vtdaaeqhpt ttaegileia ktmktklqrv htknycalkk181 kenpsftdek cknn*194 (SEQ ID NO: 22)OspC amino acids 22-210 of B.b. B31 strain (GenBank NP_047005)                        22 sgkdgntsa nsadesvkgp nlteiskkit dsnavllavk 61 eveallssid eiaakaigkk ihqnngldte nnhngsllag ayaistlikq kldglknegl121 kekidaakkc setftnklke khtdlgkegv tdadakeail ktngtktkga eelgklfesv181 evlskaakem lansvkelts pvvaespkkp*210 (SEQ ID NO: 23)LOCUS GU182319 171 bp DNA linear SYN 09-FEB-2010 DEFINITIONSynthetic construct immunodominant VlsE protein (Vs1E-d1) gene,partial cds. ACCESSION GU182319 VERSION GU182319.1 GI: 288189225translation = ″ADAAEQDGKKPEEAKNPIAAAIGDKDGDAEFNQDDMKKDDQIAA                       AIALRGMAKDGK″ (SEQ ID NO: 24) ORIGIN  1 gccgacgccg ccgagcagga cggcaagaag cccgaggagg ccaagaaccc catcgccgcc 61 gccatcggcg acaaggacgg cgacgccgag ttcaaccagg acgacatgaa gaaggacgac121 cagatcgccg ccgccatcgc cctgcgcggc atggctaagg atggaaagtg a (SEQ ID NO: 25)LOCUS GU182320 483 bp DNA linear SYN 09-FEB-2010 DEFINITIONSynthetic construct immunodominant VlsE protein (Vlse-d2) gene,partial cds. ACCESSION GU182320 translation =GAGKLFGKAGAAAHGDSEAASKAAGAVSAVSGEQILSAIVTAADAAEQDGKKPEEAKNPIAAAIGDKDGGAEFGQDEMKKDDQIAAAIALRGMAKDGKFAVKDGEKEKAEGAIKGAAESAVRKVLGAITGLIGDAVSSGLRKVGDSVKAASKETPPALNK″(SEQ ID NO: 26) ORIGIN  1 ggtgccggta agttgttcgg taaggctggt gccgcagcac acggtgatag tgaagccgcc 61 tccaaggctg ccggtgctgt aagcgctgtc tccggtgaac aaatcttgtc cgctatagtt121 accgctgccg atgcagctga acaagatggt aaaaagccag aagaagcaaa gaatcccatt181 gctgctgcaa ttggtgataa ggatggtggt gccgaatttg gtcaagatga aatgaaaaag241 gatgatcaaa tcgcagccgc catcgccctt cgcggtatgg ccaaagatgg taaatttgca301 gttaaggatg gtgaaaaaga aaaagctgaa ggcgcaatta agggtgccgc tgaaagcgcc361 gtacgcaagg tactcggtgc aattacgggt ctcattggtg atgcagtgag ctcaggtctg421 cgcaaggtgg gtgatagtgt gaaagctgcc agcaaagaaa caccccccgc cctcaacaag481 tag (SEQ ID NO: 27) LOCUS ADA82861 192 aa linear BCT 27-JAN-2010DEFINITION VlsE [Borrelia burgdorferi]. ACCESSION ADA82861 VERSIONADA82861.1 GI:282555520 DBSOURCE accession GQ506415.1 KEYWORDS . SOURCEBorrelia burgdorferi (Lyme disease spirochete)  1 egaikevsel ldklvkavkt aegassgtaa igevvadada akvadkasvk giakgikeiv 61 eaaggseklk avaaakgenn kgagklfgka gaaahagdse aaskaagavs avsgeqilsa121 ivtaadaaeq egkkpaeakn piaaaignkd ggaefgqdem kkddqiaaai alrgmakdgk181 favkednkkg ka (SEQ ID NO: 28)  1 gagggggcta ttaaggaagt tagcgagttg ttggataagc tggtaaaagc tgtaaagaca 61 gctgaggggg cttcaagtgg tactgctgca attggagaag ttgtggctga tgctgatgct121 gcaaaggttg ctgataaggc gagtgtgaag gggattgcta aggggataaa ggagattgtt181 gaagctgctg gggggagtga aaagctgaaa gctgttgctg ctgctaaagg ggagaataat241 aaaggggcag ggaagttgtt tgggaaggct ggtgctgctg ctcatgctgg ggacagtgag301 gctgctagca aggcggctgg tgctgttagt gctgttagtg gggagcagat attaagtgcg361 attgttacgg ctgctgatgc ggctgagcag gagggaaaga agcctgcaga ggctaaaaat421 ccgattgctg ctgctattgg gaataaagat gggggtgcgg agtttggtCa ggatgagatg481 aagaaggatg atcagattgc tgctgctatt gctttgaggg ggatggctaa ggatggaaag541 tttgctgtga aggaggataa taagaaaggg aaggct (SEQ ID NO: 29)LOCUS AAN87831 174 aa linear BCT 07-APR-2003 DEFINITIONvls recombination cassette Vls7 [Borrelia garinii]. ACCESSION AAN87831VERSION AAN87831.1 GI:29075690 DBSOURCE accession AY100633.1 SOURCEBorrelia garinii ORIGIN  1 asaatgnaai gdvvngdvak akggdaasvn giakgikgiv daaekadake gklnaagaeg 61 ttnadagklf vknagnvgge agdagkaaaa vaavsgeqil kaivdaakdg gekqgkkaad121 atnpidaaig gtndndaaaa fatmkkddqi aaamvlrgma kdgqfalkda aaah(SEQ ID NO: 30) ORIGIN  1 gcaagtgctg ctactggtaa tgcagcgatt ggagatgttg ttaatggtga tgtggcaaaa 61 gcaaaaggtg gtgatgcggc gagtgttaat gggattgcta aggggataaa ggggattgtt121 gatgctgctg agaaggctga tgcgaaggaa gggaagttga atgctgctgg tgctgagggt181 acgactaacg cggatgctgg gaagttgttt gtgaagaatg ctggtaatgt gggtggtgaa241 gcaggtgatg ctgggaaggc tgctgctgcg gttgctgctg ttagtgggga gcagatatta301 aaagcgattg ttgatgctgc taaggatggt ggtgagaagc agggtaagaa ggctgcggat361 gctacaaatc cgattgacgc ggctattggg ggtacaaatg ataatgatgc tgctgcggcg421 tttgctacta tgaagaagga tgatcagatt gctgctgcta tggttctgag gggaatggct481 aaggatgggc aatttgcttt gaaggatgct gctgctgctc at (SEQ ID NO: 31)LOCUS AY100628 606 bp DNA linear BCT 18-MAR-2003 DEFINITIONBorrelia afzelii strain ACAI vls recombination silent cassettelocus, complete sequence. ACCESSION AY100628 REGION: 1..606translation = ″ESAVDGVSKWLEEMIKAAKEAATKGGTGGGSEKIGDVGAANNQGAVADKDSVKGIAKGIKGIVDAAGKAFGKDGNALTGVKEVADEAGANEDAGKLFAGNAGNAAAADIAKAAGAVTAVSGEQILKAIVDGAGGAAQDGKKAAEAKNPIAAAIGADAAGA                     DFGDDMKKSDKIAAAIVLRGVAKSGKFAVANAAKKESVKSAV″(SEQ ID NO: 32) ORIGIN  1 gagagtgctg ttgatggggt tagcaagtgg ttagaagaga tgataaaagc tgctaaggag 61 gctgctacaa agggtggtac tggtggtggt agcgaaaaga ttggggatgt tggtgctgct121 aataatcagg gtgctgtagc tgataaggac agtgttaagg ggattgcgaa ggggataaag181 gggattgttg atgctgctgg gaaggctttt ggtaaggatg gtaatgcgct gacaggtgta241 aaagaagttg ctgatgaggc tggggctaac gaggatgcgg ggaagttgtt tgctggtaat301 gctggtaatg ctgctgctgc tgacattgcg aaggcggctg gtgctgttac tgcggttagt361 ggggagcaga tactgaaagc tattgttgat ggtgctggtg gtgcggctca agatggtaaa421 aaggctgcgg aggctaagaa tccgattgca gctgcgattg gggctgatgc tgctggtgcg481 gattttggtg atgatatgaa gaagagtgat aagattgctg cggctattgt tttgaggggg541 gtggctaaga gtggaaagtt tgctgttgct aatgctgcta agaaggagag tgtgaagagt601 gctgtg (SEQ ID NO: 33)

We claim:
 1. A composition comprising an isolated peptide, wherein saidisolated peptide comprises SEQ ID NO:
 1. 2. The composition of claim 1,wherein the isolated peptide consists of SEQ ID NO:
 1. 3. Thecomposition of claim 1, wherein the composition further comprises areporter polypeptide covalently linked to the isolated peptide.
 4. A kitcomprising the isolated peptide of claim
 1. 5. A composition comprisingan isolated nucleic acid encoding the isolated peptide of claim
 1. 6. Akit comprising the nucleic acid of claim 5 in appropriate packaging.